The assembly of human cytomegalovirus (HCMV) is extraordinarily complex requiring the organization and incorporation of over 100 structural proteins. The final step in assembly of the infectious particle requires acquisition of an envelope by the tegumented DNA containing capsid. In previous studies the site of envelopment has been suggested to be a membranous compartment in the cytoplasm of the infected cell, the assembly compartment (AC). From studies of the trafficking of viral glycoproteins and tegument proteins to AC, we have suggested that the AC represents a virus induced compartment at the intersection of anterograde (biosynthetic) and retrograde (endocytic) trafficking pathways marked by cellular proteins of trans-Golgi network (TGN) and the endocytic recycling compartment (ERC). In this proposal we will utilize a variety of biochemical, imaging, and genetic approaches to investigate the role of HCMV viral envelope glycoprotein trafficking through the endocytic pathway to localization in the AC and subsequently to virus assembly. In the second section of the project we will define components of the outer tegument layer of the virion and define pathways of their localization to the AC. In the final section of the proposal, w will determine requirements for production of enveloped particles, including identifying envelope glycoproteins that are essential for particle envelopment and defining interactions between envelope glycoproteins and outer tegument proteins required for virion envelopment in the cytoplasm of infected cells. These studies will provide fundamental information about HCMV assembly and identify potential targets and pathways for antiviral drug development and approaches for the production of non- replicating, virus like particle based vaccines with enhanced immunogenicity and safety profiles.